Purified regenerating retinal neurons reveal regulatory role of DNA methylation-mediated Na+/K+-ATPase in murine axon regeneration

While embryonic mammalian central nervous system (CNS) axons readily grow and differentiate, only a minority of fully differentiated mature CNS neurons are able to regenerate injured axons, leading to stunted functional recovery after injury and disease. To delineate DNA methylation changes specifically associated with axon regeneration, we used a Fluorescent-Activated Cell Sorting (FACS)-based methodology in a rat optic nerve transection model to segregate the injured retinal ganglion cells (RGCs) into regenerating and non-regenerating cell populations. Whole-genome DNA methylation profiling of these purified neurons revealed genes and pathways linked to mammalian RGC regeneration. Moreover, whole-methylome sequencing of purified uninjured adult and embryonic RGCs identified embryonic molecular profiles reactivated after injury in mature neurons, and others that correlate specifically with embryonic or adult axon growth, but not both. The results highlight the contribution to both embryonic growth and adult axon regeneration of subunits encoding the Na+/K+-ATPase. In turn, both biochemical and genetic inhibition of the Na+/K+-ATPase pump significantly reduced RGC axon regeneration. These data provide critical molecular insights into mammalian CNS axon regeneration, pinpoint the Na+/K+-ATPase as a key regulator of regeneration of injured mature CNS axons, and suggest that successful regeneration requires, in part, reactivation of embryonic signals.

The manuscript titled "Purified neuronal populations of regenerating retinal ganglion cells reveal DNA methylation-mediated role of Na+/K+-ATPase in axon regeneration" described the intrinsic properties of regenerating RGC vs non regenerating RGC. In this article author develop a new method to identify regenerative RGC and non-regenerative RGC using sciatic nerve graft. Author compares DNA methylated region of regenerative RGC to non-regenerative RGC and they identified important genes methylated during the regeneration condition. Manuscript was professionally written and well explained with few exceptions that will be improve after minor revisions Recommendation: publish after minor revision Comments 1. In introduction section line [61][62] authors discussed the role of Schwann cells and oligodendrocytes but not satellite glial cells while growing evidence suggests that Satellite Glial cell also play crucial role in axonal growth so, please add some more relevant references related to satellite glial cell such as Avraham et al. Nature Com. 2020 2. In method section, if author explain RGC tracing method in a one place, it would be good for reader. 3. Author claiming, in line [296][297], "We developed a Fluorescence-Activated Cell Sorting (FACS) ………………. following optic nerve transection in rats" in my understanding FACS sorting is normal they only develop protocol for tracing regenerative RGC using sciatic nerve graft. It would be good if author give more attention to explain the novelty of protocol. 4. I am curious about the tracer, why author only use Oregon green? they tried other tracer such as cholera toxin B and dextran. 5. In result section line 424-432, author written "Inhibition of Na+/K+-ATPase activity …………. axonal regeneration" also in text author talking about axonal regeneration while in figure 6C only present RGC number (RGC survival) not axonal regeneration, kindly use appropriate terminology (RGC survival) according to data and method. The authors in this study have developed a cell sorting method to segregate retinal ganglion cells (RGCs) and carried out WGBS profiling of these RGCs to identify genes and pathways linked to mammalian RGC regeneration. They have also reported Na+/K+-ATPase subunits play a role in embryonic and mature RGC axon growth and regeneration. However, the manuscript has multiple issues in its current form and there are some points that need to be considered to improve this work. More specific comments are given below.
• The experimental design is not clear for the entire study. The authors should include a workflow as figure 1 or in supplementary figure to explain the entire study design.
• The authors need to describe every method used in the study in detail • The authors need to mention how many male and female rat retinas were used for whole genome bisulfite sequencing for adult and embryonic stage • What was the read length for the WGBS data and please mention it in the methods • Which version of the rat genome was used for alignment for WGBS data • DMLs were called for the comparisons IR, INR, UA, and UE groups. What is IR, INR, UA, UE the authors need to define these groups in methods. • In Figure 2D the authors have explained the annotation of DMRs in the genome and majority of them are in intergenic region, but they have not mentioned in the methods how was the annotation done and what was the source of the annotation. Editor's comments:

(From Reviewer 2): In particular, we will need you to include a figure describing the experimental workflow and a clear explanation of the whole experimental set up.
Authors' Response: Thank you for the suggestion. In response, we now provide an illustrated flowchart ( Fig. 1) that describes the overall methodology to generate IR, INR, UA, and UE cells. The flowchart is accompanied by 3 videos, one for each method. Change to Text: See Videos 1-3 and Figure 1

(From Reviewer 1): Additionally, you would need to provide a more detailed description of the RGC tracing method and highlight the novelty of the method proposed in the manuscript.
Authors' Response: Done. Change to Text: Refer to answer above with regard to the RGC tracing method. Discussion: Until now, studies profiling regeneration signals in the optic nerve had relied on a comparison between injured (usually optic nerve crush or transection) and uninjured RGCs, without specifically segregating injured neurons that regenerated from those that have not. A similar FACS approach was used to separate retinal cells based on cell type (e.g., RGC vs. non-RGC) and injury state (Fischer, Petkova et al. 2004, Hartl, Krebs et al. 2017, while others separated the neurons based on their stage of development (Zibetti, Liu et al. 2019). Molecular signatures of CNS tissues that are known to regenerate after injury (frog eye and tadpole hindbrain) were compared to others that are incapable of such regeneration (e.g., frog hindbrain) (Reverdatto, Prasad et al. 2022). And more recently, RGC sorting was done by profiling cells based on genome-wide loss-of-function screen for factors limiting axonal regeneration after optic nerve crush (Lindborg, Tran et al. 2021). However, this complex method identified genes that inhibit, but not those that enhance, axon regeneration. This and other single-cell RNA sequencing technologies and transcription factor screens have yet to provide researchers with the ability to profile cells based on their proven ability to regenerate axons in vivo, underscoring both the novelty and relative simplicity of our method (Norsworthy, Bei et al. 2017, Lindborg, Tran et al. 2021, Yang, Jian et al. 2021.

In introduction section line 61-62 authors discussed the role of Schwann cells and oligodendrocytes but not satellite glial cells while growing evidence suggests that Satellite
Glial cell also play crucial role in axonal growth so, please add some more relevant references related to satellite glial cell such as Avraham et al. Nature Com. 2020 Authors' Response: We thank the reviewer for the insight and have provided to the following addition into the Introduction section of our manuscript to highlight the growing evidence of satellite glial cells in regenerative growth. Change to Text: "Moreover, a growing body of research has found that satellite glial cells, a subpopulation of glial cells found in the peripheral nervous system, promote regenerative growth in peripheral neurons following injury (Avraham, Deng et al. 2020, Jager, Pallesen et al. 2020, Avraham, Feng et al. 2021, further underscoring the importance of glial cell recruitment and function in recovery in the mammalian nervous system."

In method section, if author explain RGC tracing method in a one place, it would be good for reader.
Authors' Response: See response above. Change to Text: Flowchart (Fig. 1) and Videos 1-3, as above. 296-297, "We developed a Fluorescence-Activated Cell Sorting (FACS) ………………. following optic nerve transection in rats" in my understanding FACS sorting is normal they only develop protocol for tracing regenerative RGC using sciatic nerve graft. It would be good if author give more attention to explain the novelty of protocol.

Author claiming, in line
Authors' Response: Details and comment on novelty were added to the text. Change to Text: See Results (Paragraph 1) and Conclusions (Paragraph 1): Until now, studies profiling regeneration signals in the optic nerve had relied on a comparison between injured (usually optic nerve crush or transection) and uninjured RGCs, without specifically segregating injured neurons that regenerated from those that have not. A similar FACS approach was used to separate retinal cells based on cell type (e.g., RGC vs. non-RGC) and injury state (Fischer, Petkova et al. 2004, Hartl, Krebs et al. 2017, while others separated the neurons based on their stage of development (Zibetti, Liu et al. 2019). Molecular signatures of CNS tissues that are known to regenerate after injury (frog eye and tadpole hindbrain) were compared to others that are incapable of such regeneration (e.g., frog hindbrain) (Reverdatto, Prasad et al. 2022). And more recently, RGC sorting was done by profiling cells based on genome-wide loss-offunction screen for factors limiting axonal regeneration after optic nerve crush (Lindborg, Tran et al. 2021). However, this complex method identified genes that inhibit, but not those that enhance, axon regeneration. This and other single-cell RNA sequencing technologies and transcription factor screens have yet to provide researchers with the ability to profile cells based on their proven ability to regenerate axons in vivo, underscoring both the novelty and relative simplicity of our method (Norsworthy, Bei et al. 2017, Lindborg, Tran et al. 2021, Yang, Jian et al. 2021.